In vitro cultivation of cells, particularly eukaryotic cells, and more specifically mammalian cells, often requires special culture media that make available the growth nutrient substances that are required for efficient growth of the cells. For the efficient production of biological products from these cultured cells, including viruses or recombinant proteins, it is important that an optimal cell density is achieved as well as an increase in protein expression to obtain maximal product yield.
Cell culture media provide the nutrients necessary to maintain and grow cells in a controlled, artificial and in vitro environment. Characteristics and composition of the cell culture media vary depending on the particular cellular requirements. Relevant culture parameters include osmolarity, pH, and nutrient formulations. Cell culture media formulations have been supplemented with a range of additives, including undefined components like fetal calf serum (FCS), several animal derived proteins and/or protein hydrolysates of bovine origin.
In general, serum or serum-derived substances, such as albumin, transferrin or insulin, may contain unwanted agents that contaminate the cell cultures and the biological products obtained therefrom. For example, human serum derived additives have to be tested for all known viruses, including hepatitis and HIV, that can be transmitted by serum. Moreover, bovine serum and products derived therefrom bear the risk of bovine spongiform encephalopathy (BSE) contamination. In addition, all serum-derived products can be contaminated by unknown constituents. In the case of serum or protein additives that are derived from human or other animal sources in cell culture, there are numerous problems (e.g., the varying quality in composition of the different batches and the risk of contamination with mycoplasma, viruses or BSE), particularly if the cells are used for production of drugs or vaccines for human administration.
Many attempts have been made to provide efficient host systems and cultivation conditions, which do not require serum or other animal protein compounds. Simple serum free medium typically includes basal medium, vitamins, amino acids organic or inorganic salts, and optionally additional components to make the medium nutritionally complex.
Soy hydrolysates are known to be useful for fermentation processes and can enhance the growth of many organisms, yeasts and fungi. WO 96/26266 describes that papaic digests of soy meal are a source of carbohydrate and nitrogen and many of the components can be used in tissue culture. Franek et al. (Biotechnology Progress 16:688-692, 2000) describe growth and productivity promoting effects of defined soy hydrolysate peptide fractions.
WO 96/15231 discloses serum-free medium composed of the synthetic minimal essential medium and yeast extract for propagation of vertebrate cells and virus production process. A medium formulation composed of a basal cell culture medium comprising a rice peptide and an extract of yeast and enzymatic digest thereof, and/or a plant lipid for growth of animal cells is disclosed in WO 98/15614. A medium comprising purified soy hydrolysate for the cultivation of recombinant cells is disclosed in WO 01/23527. WO 00/03000 discloses a medium that comprises a soy hydrolysate and a yeast extract, but also requires the presence of recombinant forms of animal proteins, such as growth factors.
EP 0481791 describes a biochemically defined culture medium for culturing engineered CHO cells, which is free from protein, lipid and carbohydrate isolated from an animal source, further comprising a recombinant insulin or insulin analogue, 1% to 0.025% w/v papain digested soy peptone and putrescine. WO 98/08934 describes a serum-free eukaryotic cell culture comprising hydrolyzed soy peptides (1-1000 mg/L), 0.01 to 1 mg/L putrescine and a variety of animal-derived components, including albumin, fetuin, various hormones and other proteins. In this context, it should be also noted that putrescine is also known to be contained in standard media like DMEM/Ham's F12 in a concentration of 0.08 mg/L.
Often cell culture media may comprise trace amounts of contaminants such as detergents or preservatives which are used to prepare the components of the media. Surfactants and detergents such as N-oxides of lipid amines, Triton-X, Nonidet P40, sodium dodecyl sulfate (SDS), CHAPS, and polysorbate, to name a few, are commonly used in biochemical processes to wash solutions, membranes, glassware and many processes relied upon in recombinant biology and in biochemistry. For example, the nonionic detergent N,N-dimethyldodecylamine-N-oxide (DDAO) (CH3(CH2)11N(O)(CH3)2) (CAS number: 1643-20-5) or related compounds, alkylamine-N-oxides, are used in many areas, including cosmetics, genetic engineering, and for the study of protein membranes (Brycki et al., Polish Journal of Environmental Studies 14:411-15, 2005). DDAO is also used in the manufacturing of soy hydrolysate compounds, which are common additives in serum free media. Trace amounts of DDAO could then be detected in cell culture media that have been supplemented with contaminated soy hydrolysates. It is typically held that these trace amounts of contaminants do not effect the growth or viability of cells in culture, however, Brycki et al. (supra) studied the effects of DDAO on the denitrification (conversion of NO3− to N2) of Bacillus bacteria in culture, and showed that DDAO levels above 75 parts per million slowed the process of denitrification. However, no analysis of the effects of these trace contaminants such as DDAO in mammalian cell culture has been performed to date.
Thus there remains a need to determine the effects of culture media contaminants of cell proliferation in order to increase the growth rate of cells, and to provide an optimal cell culture medium for production of biological products, such as those used as pharmaceuticals or vaccines in humans.